Compositions and methods for treating cancer and persistent viral infections

ABSTRACT

The methods and compounds disclosed herein are useful in treating a subject having cancer or a viral infection by modulating the innate and adaptive immune systems typically by both inhibiting the function of inhibitory receptors and enhancing activity of activating receptors. Preferred therapeutic compositions comprise a carrier; at least one agent selected from the group consisting of: an anti-inflammatory agent, a cytotoxic T cell proliferation agent, or a NK cell proliferation agent; and a therapeutic peptide of the invention. In certain embodiments the compositions further include a second therapeutic peptide and/or an immunoglobulin admixed therewith in an amount sufficient to enhance passive immunoprotection in the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/US2015/039555, filed on Jul. 8, 2017, which is acontinuation-in-part of and claims priority to U.S. patent applicationSer. No. 14/697,240 filed on Apr. 27, 2015 (published as 20150299255),the contents of each of which are incorporated herein by reference intheir entireties.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readableamino acid sequence listing submitted concurrently herewith andidentified as follows: One 2,258 byte ASCII (text) file named “Seq_List”created on Jul. 8, 2015.

FIELD OF THE INVENTION

The present invention is directed to therapeutic peptides and their usesin modulating the innate and adaptive immune systems in a subject fortreatment of infectious diseases and cancers.

BACKGROUND

Viral infections pose challenges for effective treatment. While anantiviral treatment may appear to treat the initial acute infection,physical symptoms of infection often return later as persistentinfections. A common characteristic of persistent infections is thevirus' ability to successfully modulate the immune response to avoidspecific and non-specific immune defenses. In essence, persistent viralinfections are immunosuppressive diseases. In general, the course ofthese diseases is moderated by the strength of the immune system.Persistent viral infections are also highly correlated with thedevelopment of cancer.

HIV-1 is an example of a virus that causes persistent infections. HIV isperhaps the most widely known of the viruses that causeimmunosuppressive diseases. Although the immune system effectivelyproduces antibodies against these viruses in the acute stage of theinfection, the antibodies are largely non-neutralizing and allow theinfection to progress to the chronic and eventually fatal stages.Moreover, the cytolytic components of the immune system fail to destroyinfected cells even though the cells express pathogen-inducedcell-surface antigens [1]. The primary therapy against such infectionsis daily administration of a combination of anti-retroviral drugs thatinhibit viral replication after entry into the cell and subsequentmaturation. The most commonly used are nucleoside reverse transcriptaseinhibitors, non-nucleoside reverse transcriptase inhibitors, integraseinhibitors, and protease inhibitors that block enzymatic processing ofviral products. These drugs effectively inhibit replication of the virusinside an infected cell and reduce viral load in the blood toundetectable levels [2,3].

A particularly confounding aspect of infections by these viruses is theestablishment of latent reservoirs in which the integrated provirusstage can remain dormant for long periods of time. Consequently, thevirus cannot be completely cleared from an infected individual bycurrent treatments. Upon discontinuation of anti-retroviral treatment,these reservoirs are activated and the virus “rebounds” to pretreatmentlevels within a few weeks [3,4]. The question of whether the provirus isindeed dormant or simply replicates at a very low level has not beencompletely resolved. Often the immune system maintains suppression ofviral replication but fails to maintain health when the immune system iscompromised.

A defining characteristic of acquired immunodeficiency syndrome (AIDS)is the development of Kaposi sarcoma (KS), a type of cancer that affectscells lining the lymph and blood vessels. KS is caused by herpes virusin immunocompromised subjects.

Like HIV, cytomegalovirus (CMV) cause persistent infections associatedwith the immune system. CMV, however, is dormant in health individualsand generally becomes active when the immune system is compromised. Theantiviral drug ganciclovir, a viral DNA polymerase inhibitor, iscommonly used to treat acute cytomegalovirus (CMV) infections. Human CMVhas also been found to play a role in the development of cancer throughoncomodulation, e.g., enabling cancer cells to evade immune recognitions[44].

Chronic viral hepatitis is the most common risk factor worldwide forliver cancer. Like HIV, Hepatitis C virus (HCV) is a RNA virus and ismore likely to result in chronic infection than hepatitis B virus (HBV).Recent advances in the treatment of HCV involve development of proteaseinhibitors that act in a similar manner as those used to treat HIV-1infections [5]. For HCV, the protease inhibitors are added to thecurrently accepted drug regime of pegylated interferon-alpha andribavirin.

About one-third of the world's population has evidence of a hepatitis Binfection, either current or past, which is more than HIV and HCVinfections combined [6]. Most healthy adults raise an effective immuneresponse against hepatitis B virus (HBV), but the effectiveness of theimmune defense is dependent upon the activity of natural killer (NK)cells [6]. NK T cells contribute to resolution of a HBV infection, withthe NKG2D receptor playing a key role [6]. HBV establishes a chronicinfection, and although infected cells express the hepatitis B surfaceantigen (HBsAg), the immune system is unable to prevent progression ofthe infection. Long term continuing virus replication leads toprogression to cirrhosis and hepatocellular carcinoma [6-8]. Infectionby HBV is the leading cause of hepatocellular carcinoma. Approximately662,000 deaths occur worldwide each year, with roughly half of them inChina [9].

A potentially powerful therapeutic approach for these persistent viralinfections and immunosuppressive diseases is a combination of drugs withantiviral activity, in particular those that bind to NKG2D, and thosewith strong anticancer activity that promote induction of proliferationof activated cells of the innate and adaptive immune system.

An Alternative Approach to Therapy

In contrast to therapeutic approaches aimed at prevention or control ofdisease by directly inhibiting a step in the viral replication cycle, asdescribed above, or by the use of highly toxic cytotoxicchemotherapeutic drugs for cancer treatment, reactivation of patients'immune system is an alternative therapy that holds promise for restoringhealth and productivity to an infected patient in a practical,cost-effective manner. This approach provides a general defense againstdiseases rather than a pathogen-specific treatment. As a result, anintense interest in immunotherapy, as indicated by the development ofcytokine and monoclonal antibody treatments, is leading to products thatcan stimulate or inhibit the immune system.

The role of cytokines in the inhibition of HIV infectivity, particularlyinterleukin-16 (IL-16), interleukin-8 (IL-8) and RANTES (Regulated uponActivation, Normal T-cell Expressed, and Secreted; also known as CCL5),is very important. Cytokines such as IL-16, IL-8 and RANTES, which haveoverlapping and complementary functions, can act to attenuate viralinfection by competing with viral binding with the receptors and byinterfering with viral entry into cells by down-regulating the receptorsrequired for entry. Other cytokines such as interferons (e.g., IFN-α.and IFN-γ) act to reduce viral load by activating intracellularanti-viral enzymes and also by stimulating antibody-mediatedphagocytosis. These cytokines have also been shown to be effective inthe acute stage of HBV infection [6].

Interleukins (IL's) and interferons (IFN's) are potent cellularstimulants that are released from a variety of cells in response toinsult or injury. Consequently, these proteins have attracted intenseinterest as therapeutic agents. IL-16 is a natural ligand of CD4 andshould compete with virus for binding to T cells. IL-21 is required toavoid depletion of CD8⁺ T cells and also essential to maintain immunityand resolve persistent viral infections [10-12]. Similar to generalstimulants such as lipopolysaccharide (LPS), however, IL's and IFN'sinduce release of inflammatory cytokines. Therefore, when given athigher than normal endogenous concentrations, they often havesubstantial adverse effects, which can be life threatening and mayrequire inpatient treatment facilities. Similarly, levels of TNF-α,IL-1β and IL-6 are directly correlated with the probability of death inhumans. Moreover, production of recombinant IL's and IFN's and theirapplication are very costly. Even lower-dosage immunostimulanttreatments developed for out-patient use have lower success rates andare not suitable in some situations such as, for example, to extendremission from cancer therapy or control a disease such as HIV at achronic level. In view of this, it appears that exogenous therapeuticagents such as large, intact cytokine molecules are not well suited forgeneral therapeutic use.

Usually, infections are cleared by the immune system through (i)internalization of the pathogen and presentation of antigens to T and Bcells by dendritic cells (DCs), (ii) generation of antibodies by Bcells, (iii) lysis of pathogen-infected cells by NK cells and CD8⁺cytotoxic T lymphocytes (CTL), and/or (iv) destruction of the virus orcancer cell by antibody-mediated phagocytosis. While neutralizingantibody responses are subject to pathogen escape, many non-neutralizingantibodies that nevertheless bind the pathogen are present in infectedpatients. Restoration of immune effector cell functions, in particularphagocytic activity, which can recognize the resulting antigen-antibodycomplexes and destroy the complexes by antibody (Fc)-mediatedphagocytosis, may be applicable to the clearance of infections ingeneral.

The cell types that have significant involvement in viral infections inaddition to phagocytic cells are in particular, two subsets of the Tcell population (CD3⁺ and CD8⁺), NK cells (CD56⁺) and CTLs (CD8⁺). Thesecells are able to kill virus-infected cells and cancer cells byantibody-dependent cellular cytotoxicity (ADCC) in addition to anability to directly lyse infected cells. NK cells are an integralcomponent of the innate immune system and are primarily responsible forkilling virus-infected and cancer cells. NK cells and CTL kill theirtargets mainly by releasing cytotoxic molecules such as perforin,granzymes and granlysin, which are contained in intracellular granules.These molecules are released when these cells make contact with targetcells that contain antigens on the surface of viral infected or cancercells to which antibodies bind. Activated NK cells also releasecytokines and chemokines such as IFN-γ that activates macrophages anddrives differentiation of CD4⁺ T cells into type 1 (Th1) cells [11,12].

Information relevant to attempts to address one or more of theseproblems can be found in the following references: U.S. PatentPublication No. 2007/0003542; U.S. Patent Publication No. 2006/0269519;U.S. Patent Publication No. 2004/0248192; P. W. Latham, 1999;Fatkenheuer et al., 2005; Stover et al., 2006; Cohen, 2007;GlaxoSmithKline, 2005a and GlaxoSmithKline, 2005b. Each one of thesetreatments referred to in these references, however, suffers from one ormore of the following disadvantages:

1. the size or composition of the agent provides significant challengesto cost-effective synthesis and purification;

2. the agent is specific for particular pathogen and/or cell type,rendering them unsuitable for general therapeutic use;

3. treatment with the agent induces clinically deleterious side effectsthat can be life-threatening, such as inflammation or hepatotoxicity,and require inpatient treatment facilities;

4. termination of treatment is followed soon thereafter by an increasedsystemic viral load;

5. long term exposure to agent often leads to treatment-resistantpathogens;

6. lower-dosage treatments developed for out-patient use have lowersuccess rates and are not suitable in some situations;

7. treatment is ineffective, impractical, or cost-prohibitive for alarge proportion of patients;

8. development of therapeutic antibodies require considerable medicalinfrastructure;

9. treatment such as vaccines may be appropriate to prevent infectionbut not to treat those already infected and who have a suppressed immunesystem;

10. no beneficial synergy between the immunogenic response induced andthe effects of other endogenous immunoregulators;

11. agent inhibits the release of inhibitory cytokines that suppressrelease of beneficial cytokines, an indirect treatment; and

12. agent acts to restore baseline cytokine levels to balance responsesof the immune system rather than promoting activation of phagocytes.

Many of these therapeutic protocols also become ineffective with timebecause mutation of the pathogen allows it to escape the treatment.Moreover, any immunosuppression that accompanies the disease attenuatesthe ability of the innate and adaptive immune systems to respond toantigenic changes and thereby keep the infection under control.

The immune system in individuals infected with a pathogenic agents suchas HIV-1 or HBV initiate a defense response by production of antibodies.Even though the virus may mutate at one or a few sites and therebyescape the neutralizing activity of antibodies, endogenously producednon-neutralizing antibodies are usually polyclonal and may still bindthe virus. The presence of anti-viral antibodies is often used as adiagnostic test for infection. During the course of the disease, thecellular components of the innate and adaptive immune response thenbecome absent or quiescent. When the immune defense mechanisms reach asufficiently low level, viral replication is not held in check andrapidly leads to a final stage of the disease. However, even at thislate stage, patients can be rescued from death by aggressive therapy.Therefore, an agent that reactivates cells of the immune system, inparticular phagocytes and NK cells, will likely restore an immunedefense against progression of the disease.

Not only is it essential to overcome the suppressive mechanisms of thepathogen, it is also important to modulate the host's natural mechanismthat suppress the immune system. Therapeutic agents thatactivate/reactivate the immune system show particular promise in thisregard, including cytokines and immunomodulators, although therapiesbased on exogenous agents such as large, intact cytokine molecules arenot generally well suited for therapeutic use. Peptides, however, areoften much more suitable therapeutic agents than large polypeptides orproteins. Peptides can, for example, be designed to induce one or moreparticular desired effects in vitro or in vivo, often withoutconcomitantly inducing deleterious effects, and can usually besynthesized in a cost effective manner.

The development of this technology is applicable to diseases caused byviruses, bacteria, fungi and cancers.

SUMMARY OF THE INVENTION

The present invention is directed to methods of treating animmunocompromised subject, the method comprising: administering to theimmunocompromised subject a composition comprising a therapeutic peptideor a multivalent structured polypeptide comprising multiple copies ofthe therapeutic peptide, the therapeutic peptide consisting of 5 to 8amino acids and selected from the group consisting of:

VGGGS (SEQ ID NO:1) and

X1-X2-X3-X4-X5-X6-X7-X8,

wherein:

X1 is selected from the group consisting of H and N;

X2 is selected from the group consisting of P and Q;

X3 is selected from the group consisting of S and H;

X4 is selected from the group consisting of H, T, and L;

X5 is selected from the group consisting of P and K, or is absent;

X6 is selected from the group consisting of R, L and S, or is absent;

X7 is selected from the group consisting of S and L, or is absent; and

X8 is G, or is absent;

wherein the therapeutic peptide or multivalent structured polypeptide isin an amount sufficient to increase, activate, and/or stimulateproliferation of immune cells in the subject.

The present invention is also directed to methods of treating a subjecthaving cancer and/or a viral infection: the method comprisingadministering to the subject with cancer and/or a viral infection atherapeutic peptide or a multivalent structured polypeptide comprisingmultiple copies of the therapeutic peptide, the therapeutic peptideconsisting of 5 to 8 amino acids and selected from the group consistingof:

VGGGS (SEQ ID NO:1) and

X1-X2-X3-X4-X5-X6-X7-X8,

wherein:

X1 is selected from the group consisting of H and N;

X2 is selected from the group consisting of P and Q;

X3 is selected from the group consisting of S and H;

X4 is selected from the group consisting of H, T, and L;

X5 is selected from the group consisting of P and K, or is absent;

X6 is selected from the group consisting of R, L and S, or is absent;

X7 is selected from the group consisting of S and L, or is absent; and

X8 is G, or is absent;

wherein the therapeutic peptide or multivalent structured polypeptide isin an amount sufficient to treat the cancer and/or virus by increasingproliferation of immune cells in the subject.

In certain specific embodiments of the aforementioned methods,therapeutic peptide may be selected from the group consisting of one ormore of the following: VGGGS (SEQ ID NO:1), HPSLK (SEQ ID NO:2),NPSHPLSG (SEQ ID NO:3), NPSHPSLG (SEQ ID NO:4), and NQHTPR (SEQ IDNO:5). In some implementations, the subject may have a persistent viralinfection, which may be selected from the group consisting of: anHIV/AIDS infection, a CMV infection, a HBV infection, and a HCVinfection.

In a particular non-limiting embodiment, the invention is directed to amethod of treating a subject having cancer by administering to thesubject with cancer a therapeutic peptide or a multivalent structuredpolypeptide comprising multiple copies of the therapeutic peptide,wherein the therapeutic peptide is NQHTPR (SEQ ID NO:5), wherein thetherapeutic peptide or multivalent structured polypeptide is in anamount sufficient to treat the cancer in the subject.

In one embodiment, the multivalent structured polypeptide isadministered to the subject and is branched. In certain non-limitingaspects of the invention the therapeutic peptide or multivalentstructured polypeptide administered to the subject stimulatesproliferation of immune cells selected from the group consisting ofmacrophages; dendritic cells; natural killer cells; natural killer Tcells; CD3+, CD4+ and CD8+ T cells; B cells; and combinations thereof.

In a specific embodiment, the therapeutic peptide or multivalentstructured polypeptide is administered in an amount sufficient to induceantibody-mediated cellular cytotoxicity in the subject, preferably toincrease the expression of at least one endogenous cytokine fromlymphocytes elected from the group consisting of: IL-2, IL-4, IL-12,IL-16, IL-17, IL-21, TNF-β, IFN-γ and RANTES and/or decreases at leastone endogenous cytokines elected from the group consisting of: IL-lα,IL-1β, IL-13, and TNF-α. In addition, the multivalent structuredpolypeptide induces rapid modifications of phosphorylated kinasesinvolved in signal transduction to achieve these activities.

In certain implementations, the present method further comprises thestep of determining (a) the level of immune cells in theimmunocompromised subject before administering the composition; and (b)the level of immune cells in the immunocompromised subject afteradministering the composition. The levels of (a) and (b) may bedetermined with flow cytometry. Examples of preferred ratios of (b) to(a) is at least 2, at least 3, at least 4, or at least 5. Yet otheraspects of the invention, the ratio of (b) to (a) is at least 1.5, atleast 2, at least 2.5, at least 3, at least 3.5, at least 4, at least4.5, or at least 5.

In some implementations of the present invention, the therapeuticpeptide preferably functionally mimics a terminal sequence5-acetylneuraminic acid-galactose on complex glycans or glycoproteins,the terminal sequence being linked α(2,3) or α(2,6). The therapeuticpeptide preferably functionally mimics a terminal N-acetylgalactosamineor galactose on cell-surface glycoproteins. The therapeutic peptides areadvantageously configured to bind to the receptor NKG2D and/or siglecsand function as modulators of the immune system by binding to receptorson B cells, DCs, NK cells, T cells, cytotoxic T cells and/or phagocyticcells. In addition, therapeutic peptides bind to receptors specific forN-acetylgalactosamine or galactose such as CLEC10a/CD301 on immature DCsand macrophages, langerin on DCs and asialylglycoprotein receptor-1 onhepatic cells. In some embodiments, the therapeutic peptide ormultivalent structured polypeptide activates the immune cells by bindingto the inhibitory siglec receptors. In some embodiments, the therapeuticpeptide or multivalent structured polypeptide activates the immune cellsby binding to an activating receptor including NKG2D and/or CLEC10a.

The methods of the present invention may further comprise administeringto the subject a second therapeutic peptide or a multivalent structuredpolypeptide comprising multiple copies of the second therapeuticpeptide. The second therapeutic peptide consists of 5 to 8 amino acidsand selected from the group consisting of:

VGGGS (SEQ ID NO:1); and

X1-X2-X3-X4-X5-X6-X7-X8,

wherein

X1 is selected from the group consisting of H and N;

X2 is P;

X3 is S;

X4 is selected from the group consisting of H and L;

X5 is selected from the group consisting of P and K, or is absent;

X6 is selected from the group consisting of L and S, or is absent;

X7 is selected from the group consisting of S and L, or is absent; and

X8 is G, or is absent;

In some implementations, the second therapeutic peptide is selected fromthe group consisting of: VGGGS (SEQ ID NO:1), HPSLK (SEQ ID NO:2),NPSHPLSG (SEQ ID NO:3), and NPSHPSLG (SEQ ID NO:4). In some aspects, themultivalent structured polypeptide comprising the second therapeuticpeptide is administered to the subject, wherein the multivalentstructured polypeptide is branched. In some implementations, the secondtherapeutic peptide functionally mimics a terminal sequence5-acetylneuraminic acid-galactose or N-acetylgalactosamine on complexglycans, the terminal sequence being linked α(2,3) or α(2,6) or terminalsugars such as N-acetylgalactosamine or galactose.

The therapeutic peptide or multivalent structured polypeptide of theaforementioned methods may be in a composition comprising a carrier. Insome embodiments, the composition may further comprises at least oneagent selected from the group consisting of: an anti-inflammatory agent,a cytotoxic T cell proliferation agent, or a NK cell proliferationagent; and a therapeutic peptide or a multivalent structuredpolypeptides of the invention. Such compositions may further comprise anantibody preparation admixed in an amount sufficient to enhanceantibody-mediated cellular cytotoxicity in a subject; or animmunoglobulin admixed therewith in an amount sufficient to enhancepassive immunoprotection in the subject

Therapeutic compositions are also contemplated in the present invention.The therapeutic compositions of the present invention comprise acarrier, a therapeutic peptide or a multivalent structured polypeptidecomprising multiple copies of the therapeutic peptide, wherein thetherapeutic peptide is NQHTPR (SEQ ID NO:5), wherein the therapeuticpeptide or multivalent structured polypeptide is in an amount sufficientto treat the cancer in a subject; and at least one agent selected fromthe group consisting of: a B cell proliferative agent, a cytotoxic Tcell proliferation agent, or a NK cell proliferation agent. Thecomposition may further comprise an antibody preparation admixed in anamount sufficient to enhance antibody-mediated cellular cytotoxicity orfurther comprises an immunoglobulin admixed with the polypeptidecomposition in an amount sufficient to enhance passive immunoprotection.

In some embodiments, the therapeutic composition further comprises asecond therapeutic peptide or a multivalent structured polypeptidecomprising multiple copies of the second therapeutic peptide. The secondtherapeutic peptide consists of 5 to 8 amino acids and selected from thegroup consisting of:

VGGGS (SEQ ID NO:1); and

X1-X2-X3-X4-X5-X6-X7-X8,

wherein

X1 is selected from the group consisting of H and N;

X2 is P;

X3 is S;

X4 is selected from the group consisting of H and L;

X5 is selected from the group consisting of P and K, or is absent;

X6 is selected from the group consisting of L and S, or is absent;

X7 is selected from the group consisting of S and L, or is absent; and

X8 is G, or is absent;

In some aspects, the second therapeutic peptide is selected from thegroup consisting of: VGGGS (SEQ ID NO:1), HPSLK (SEQ ID NO:2), NPSHPLSG(SEQ ID NO:3), and NPSHPSLG (SEQ ID NO:4). In some implementations, themultivalent structured polypeptide comprising the second therapeuticpeptide is administered to the subject, wherein the multivalentstructured polypeptide is branched.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 depict models of monovalent therapeutic peptides.

FIG. 1 illustrates a model of monovalent SVH1C (SEQ ID NO:3,space-filled structure) docked in the glycan-binding site of thereceptor Siglec-5 (accession no. 2ZG1), predicted binding energy,ΔG′=−47 kJ/mol. The predicted binding energy suggests strong interactionwith the receptor, with a K_(D)=˜1×10⁻⁸ M.

FIG. 2 illustrates a model of monovalent SVH1C (SEQ ID NO:3,space-filled structure) docked in the ligand binding site of thereceptor NKG2D (accession no. 1MPU). The predicted binding energy forNPSHPLSG (SEQ ID NO:3) was ΔG′=−40 kJ/mol, which corresponds to aK_(D)=˜1×10⁻⁷ M.

FIG. 3 illustrates a model of monovalent SV6D (SEQ ID NO:5, space filledstructure) docked in the ligand binding site of the receptorasialylglycoprotein receptor-1 (ASGPR-1) (accession no. 1DV8), a proteinhomologous to CLEC10a that is highly specific for N-acetylgalactosamine.The predicted binding energy was ΔG′=−40 kJ/mol, which corresponds to aK_(D)=˜1×10⁻⁷ M.

FIG. 4 illustrates a model of the final design of the peptide. Fouridentical, active sequences (e.g., arms 1 and 2) were extended from acentral core (4), composed of tri-lysineamide, by a linker sequence (3).

FIG. 5 demonstrates and shows the multivalent structure of SVH1C([NPSHPLSGGGGS]4K3-NH2). N, asparagine; P, proline; S, serine; H,histidine; L, leucine; G, glycine; K, lysine. The molecular weight ofthe peptide is 4,593.9. A linker sequence (GGGS) extends the activesequence from the tri-lysine core.

FIG. 6 demonstrates and shows the multivalent structure of SV6D([NQHTPRGGGS]4K3-NH2). N, asparagine; Q, glutamine; H, histidine; T,threonine; P, proline; R, arginine, G, glycine; S, serine; K, lysine.The molecular weight is 4,369.1. A linker sequence (GGGS) extends theactive sequence from the tri-lysine core.

FIG. 7 illustrates the binding of SVH1C (SEQ ID NO:3) to lectin-typereceptors, siglecs (sialic acid-binding Ig-like lectin receptors) andother lectin-type receptors in a solid-phase assay. The buffer in theseassays was PBS containing 0.05% Tween-20. The Figure shows the amount ofstreptavidin-peroxidase bound to SVH1C that was bound to the receptors.The receptors were Fc-chimeras and were assayed in protein A/G-coatedmicrotiter wells. Siglec-1 and CLEC10a contained a C-terminal His tagand were assayed in a separate experiment with Ni-coated wells. SEM wasdetermined from four independent experiments run in duplicate.Inhibition by fetuin is shown by the average of two assays in which theglycoprotein was added at 10 μM (second, black bar) or 30 μM (third,light grey bar) in each receptor group. Binding was measured by acolorimetric assay of peroxidase.

FIG. 8 illustrates the binding of SVH1C (SEQ ID NO:3) to receptor NKG2Din a solid-phase assay. A, The amount of streptavidin-peroxidase boundto SVH1C that was bound to the receptor was measured after extensivewashing with PBS containing 0.05% Tween-20. Fetuin (5 μM, black bar; 10μM, light grey bar; 30 μM, grey bar) and sialyllactose (12 μM, blackbar; 20 μM, light grey bar; 40 μM, grey bar) were included asinhibitors. Binding was measured by a colorimetric assay of peroxidase.This assay was performed three times. B, Graphical representation ofinhibition of binding of SVH1C to NKG2D by fetuin (circles) orsialyllactose (squares).

FIG. 9 illustrates the changes in phosphorylation of cell-surfaceimmunoreceptors after treatment of human PBMCs with 100 nM SVH1C (SEQ IDNO:3) for 5 min. Cell lysates were spread on an array of captureantibodies and phosphorylated forms were detected by a phospho-tyrosineantibody (Human Phospho-Immunoreceptor Array, R&D Systems, Minneapolis,Minn.). The phosphorylated forms of CD229, FcγRIIA, LAIR-1, Siglec-2,-3, -5, -7, -9, and -10 decreased, while BLAME (B Lymphocyte ActivatorMacrophage Expressed) increased. FcRH4, SHP-1 and NKp46 did not changesignificantly. This experiment showed that the peptide has dramaticeffects on human cells.

FIG. 10 illustrates that alternate-day subcutaneous injection of SVH1C(SEQ ID NO:3) into C57BL/6 mice resulted in relatively large increasesin populations of immune cells in the peritoneal cavity. The bars, inincreasing darkness, show populations of specific cell types at 1, 3 and5 days of treatment, i.e., 24 hours after each injection at day 0, 2 and4. Peritoneal cells were obtained from 3 animals, pooled, and analyzedby flow cytometry. The markers used to identify cell types are listedacross the top of the figure. The total number of each cell type isplotted, with the scale indicated at the top of each cell type. Cellsare identified by the usual designations across the bottom of thefigure. An asterisk indicates the activated populations that expressCD69.

FIG. 11 shows the antiviral activity of SVH1C (SEQ ID NO:3) and SV6B(SEQ ID NO:2) by inhibition of HIV-1 replication in PBMC cultureswithout anti-HIV antibodies.

FIG. 12 illustrates the binding of SV6D (SEQ ID NO:5) to lectin-typereceptors, CLEC10a, langerin, ASGPR-1, dectin-1, and other lectin-typereceptors in a solid phase assay. The buffer in these assays was PBScontaining 0.05% Tween-20. The Figure shows the amount ofstreptavidin-peroxidase bound to SV6D that was bound to the receptors.The receptors were Fc-chimeras and were assayed in protein A/G-coatedmicrotiter wells. The assay was similar to that described under FIG. 7.

FIG. 13A and FIG. 13B illustrates the changes in phosphorylation ofsignal transduction kinases in human PBMCs in culture treated with 100nM SV6D for 5, 10 or 15 min. Cell lysates were spread on an array ofcapture antibodies and phosphorylated forms were detected with a HumanPhospho-Kinase Array (R&D Systems (Minneapolis, Minn.).

FIG. 14 illustrates that alternate-day subcutaneous injection of SV6D(SEQ ID NO:5) into C57BL/6 mice resulted in relatively large increasesin populations of immune cells in the peritoneal cavity. The bars, inincreasing darkness, show populations of specific cell types at 1, 3 and5 days of treatment, i.e., 24 hours after each injection at day 0, 2 and4. Peritoneal cells were obtained from 3 animals, pooled, and analyzedby flow cytometry. The markers used to identify cell types are listedacross the top of the figure. The total number of each cell type isplotted, with the scale indicated at the top of each cell type. Cellsare identified by the usual designations across the bottom of thefigure. An asterisk indicates the activated populations that expressCD69.

FIG. 15 illustrates the effect of treatment of C57BL/6 mice implantedwith an ovarian cancer cell line. Body weights of individual mice weremeasured during treatment with 1 or 0.1 nmole/g doses of SV6D (SEQ IDNO:5) injected subcutaneously every other day. Ascites formation wasevident at the start of treatment and the weights shown are after twoweeks of treatment. The effectiveness of the 0.1 nmole/g dose wassimilar to that of the drug paclitaxel, the currently usedchemotherapeutic drug.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to compositions and methods foractivation and proliferation of macrophages, B cells, DCs, NK cells, Tcells and/or CD8+ cytotoxic T lymphocytes. The compositions and methodsinvolve the use of peptidic mimetics of glycan ligands of receptors thatcan overcome immunosuppression, produce antiviral activity, and/oranticancer activity.

Overcoming Immune System Suppression

Inhibitory mechanisms naturally maintain a balance within the immunesystem to prevent progression of an over-stimulated, deleterious immuneresponse. Major components in these inhibitory mechanisms arecell-surface, lectin-type receptors of the siglec (sialic acid-bindingimmunoglobulin-like lectins) family [13,14]. The cytoplasmic domains ofmost siglecs contain ITIMs (immunoreceptor tyrosine-based inhibitorymotifs) that when phosphorylated recruit tyrosine phosphatases such asSHP-1. Siglecs are abundant cell-surface proteins and bind to sialicacid residues on glycoprotein subunits of activating receptor complexessuch as the B cell receptor (BCR). Siglec-associated SHP-1 thendephosphorylates (deactivates) the activating complexes, whichsuppresses immune functions [13-15].

Most cells of the immune system contain several siglecs [13-15]. A totalof 14 siglecs is expressed in humans. As an example, the extensivelystudied Siglec-2 binds to sialic acid residues on BCR-associatedproteins such as IgM, causes suppression of the activity of BCR inantigen recognition and antibody production [14,15]. Siglec-2 containssix tyrosine residues within its cytoplasmic domain, three of which arewithin three ITIMs, which are potentially phosphorylated. Sialosideswith high affinity to Siglec-2 bind to the sialic acid-binding site ofSiglec-2 and release it from the BCR complex [14,16]. Consequently,activation of the BCR complex is no longer attenuated and B cellactivation ensues. It can be expected that similar suppression of otherimmune cells by siglecs is also relieved by binding of small moleculesthat inhibit the suppressive activity of these receptors. Whereas mostsiglecs express inhibitory functions, Siglec-14, -15 and -16 lackcytoplasmic ITIMs and serve activating functions in association with anadaptor protein DAP12, which contains an ITAM (immunoreceptortyrosine-based activating motif) [13-15]. A compound that binds tomultiple siglecs should provide a powerful mechanism to achieve multipleimmune cell activation by decreasing the suppressive ability of siglecswhile also increasing activating functions. An additional receptor thatbinds sialic acid-galactose sequences is the potent activating receptor,NKG2D, which is expressed on NK cells, γδ T cells and CD8⁺ cytotoxic Tcells [17,18]. NKG2D also functions with cytoplasmic adaptor proteinsDAP10 and DAP12 that contain ITAMs. One set of peptides described inthis invention are mimetics of sialic acid-galactose sequences and bindwith high avidity to these receptors [19].

The peptides of this invention that mimic sialic acid, such as NPSHPLSG(SEQ ID NO:3), have a dual function. Firstly, they bind to theinhibitory siglec receptors. Secondly, they also bind to the activatingreceptor, NKG2D. The siglecs, as inhibitory receptors that preventexcessive activation of the immune system, are ‘checkpoints’. By bindingto and inactivating these receptors, the peptides of this invention canbe considered ‘checkpoint inhibitors’. Concomitantly, by binding toactivating receptors on the same cell, the peptides exert a strongstimulatory effect on these cells. Essential for this function is themuch greater avidity of the peptides to the sialic acid-binding sitesthan natural, cell-based glycans. In fact, siglecs bind sialicacid-containing ligands with K_(D) values in the low millimolar range,whereas the peptides bind with three orders of magnitude greateravidity, with K_(D) values of 1 micromolar or less. Thus the peptidesare well suited to provide therapeutic benefit.

Thus, interaction of peptides with lectin-type inhibitory receptorsleads to activation of immune cells. These activities can be coupledwith other peptides that bind to activating receptors and directlyactivate immune cells. Lectins are generally highly-specific,carbohydrate-binding proteins, and these receptors interact with ligandsthat contain sugar residues. Cells of the immune system express anextensive array of regulatory, C-type lectin cell-surface receptors[20,21]. A second set of peptides of this invention are mimetics ofN-acetylgalactosamine (GaINAc) and galactose (Gal) and bind to receptorsthat lead to activation of immune cells. A receptor with this ligandspecificity is the macrophage galactose-type lectin (MGL), acalcium-dependent (C-type) lectin also designated CLEC10a or CD301 [22].Two forms of this receptor are expressed in the mouse, one that isspecific for galactose (Gal, MGL1) and the second specific for GaINAc(MGL2). CLEC10a is expressed by macrophages and immature dendriticcells, which are the primary antigen-presenting cells (APCs). Thelectin-type receptors on immune cells therefore present an entry intothe immune system.

CLEC10a binds to a glycan on the ubiquitous phosphatase CD45 andattenuates its activity. The phosphatase activity of CD45 is requiredfor lymphocyte activation [23]. Engaging CLEC10a with a peptide thatbinds with much higher avidity than the glycan on CD45 leads to cellularactivation. Thus, CLEC10a is a strategic target for activation of immunecells. An extensive literature has demonstrated that targeting CLEC10apromotes internalization of antigens by DCs, presentation of antigens toCD4⁺ T cells and differentiation of IFNγ-producing CD4⁺ T cells [22,24].Ligand binding to CLEC10a also results in enhanced antigen-specific,IFNγ-producing CD8⁺ T cell responses and tilts naïve CD4⁺ T cellstowards Th1 cells, with increased proliferation of T cells. In addition,DCs mediate activation and proliferation of natural killer (NK) cells[25]. The phosphatase activity of CD45, a widely expressed and abundantcell surface protein, is required for lymphocyte activation anddevelopment [23]. Trans binding of CLEC10a on DCs to a GaINAc residue onCD45 on T cells results in T cell inhibition [26]. Introduction of aGaINAc-containing factor displaces CLEC10a from CD45 and allowsdephosphorylation of inhibitory receptors and T cell activation [24].CLEC10a is the likely receptor for the macrophage activating factor, aclinically potent anti-cancer derivative of serum group specificcomponent-1 (Gc1) that contains a covalently-bound GaINAc. A hydrolaseexpressed by some cancer cells removes the GaINAc residue, therebyinactivating the protein [27]. These results suggested a critical rolefor receptors of GaINAc-containing ligands in regulation of immune cellsand the treatment of cancer.

Current immunotherapy of cancer has focused on the use of monoclonalantibodies to bind to, and inhibit the activity of, inhibitory receptorson T cells such as PD-1 and CTLA-4. Antibodies have also been developedagainst a protein, PD-L1, that is highly expressed by cancer cells andacts as an activating ligand for PD-1. Combinations of these antibodieshave proven to be very effective in treating certain types of cancer[28]. The antibodies are injected intravenously and cause significanttoxic side-effects. Although the peptides of this invention interactwith different inhibitory receptors, the overall result may be the samebut simply be achieved through different mechanisms and withconsiderably less toxicity.

Peptidic Mimetics of Glycan Ligands of Receptors

An important component of immune system stimulation by the peptides isactivation and proliferation of B cells, DCs, NK cells, T cells and CTL(cytotoxic T lymphocytes) in addition to activation of phagocytic cells.To this end, peptidic mimetics of the glycan 5-acetylneuraminicacid-galactose [Neu5Ac(α2,3)Gal and Neu5Ac(α2,6)Gal] were designed.These glycans bind to NKG2D, an important activating receptor on NKcells, γδ T cells and CD8⁺ cytotoxic T cells [18], and to the family ofsiglecs (sialic acid-binding Ig-like lectin) receptors that is presenton most cells of the immune system and are generally inhibitoryreceptors [13-15]. Whereas identified endogenous ligands of NKG2D areseveral protein-based activating ligands [17,29], binding of glycansshould also activate these cells. Activation of phagocytes occurs bybinding of peptides to siglecs or other receptors on these cells. Thetherapeutic peptides consist of a multivalent structure in which thearms consist of sequences only 9 to 12 amino acids long (including alinker sequence). The active sequences of the relevant peptides thatwere described previously [U.S. Pat. Nos. 7,811,995 and 8,496,942,incorporated by reference thereto] are VGGGS (SEQ ID NO:1), HPSLK (SEQID NO:2), NPSHPLSG (SEQ ID NO:3), and NPSHPSLG (SEQ ID NO:4).Preferably, the peptides are in substantially pure form. Typically it isdesired that the peptides be at least 70%, more preferably at least 80%,and most preferably at least 95% pure by weight. In one embodiment theN-terminus may also be acetylated.

Additionally, peptide NQHTPR (SV6D; SEQ ID NO:5) was designed to bind tostrategic receptors that are regulated by glycans containing terminalN-acetylgalactosamine or galactose. SV6D (SEQ ID NO:5) stronglystimulated proliferation and activation of immune cells in theperitoneal cavity and effectively inhibited progression of ascites in amodel of ovarian cancer. It is expected that this peptide is alsoeffective in treating cancers of other peritoneal organs. The activesequences of the relevant peptides were described previously [U.S. Pat.Nos. 7,838,497 and 8,460,697, incorporated by reference thereto].

In a preferred embodiment, the peptides of the invention comprise apeptide construct with at least two arms. The construct typically has acentral framework and each arm comprises a therapeutic sequence linkedto the central framework via a linker. Each therapeutic sequence of thepeptide construct can be the same or different. In a preferredembodiment, the therapeutic sequence is the same for each arm of peptideconstruct. The therapeutic sequence is preferably selected from thegroup of therapeutic peptides described above. The present inventionalso provides therapeutic compositions comprising at least one peptideof the invention and a pharmaceutically acceptable carrier. In apreferred embodiment, the composition is an immunostimulatorycomposition, preferably further comprising an antigen and/or an antibodypreparation admixed therewith in an amount sufficient to enhanceantibody-mediated cytotoxicity or phagocytosis. Alternatively, thecomposition may comprise an immunoglobulin admixed with the therapeuticpeptide in an amount sufficient to substantially enhance passive immuneprotection, e.g., at least 30% increase compared to the control.

In another embodiment, the therapeutic compositions comprises a carrier;at least one agent selected from the group consisting of: a B cellproliferation agent, a dendritic cell proliferation agent, a cytotoxic Tcell proliferation agent, or a NK cell proliferation agent; and atherapeutic peptide or a multivalent structured polypeptide as describedabove. In certain embodiments, the composition further comprises anantibody preparation admixed in an amount sufficient to enhance antibodymediated cellular cytotoxicity in a subject; or further comprises animmunoglobulin admixed with the polypeptide composition in an amountsufficient to enhance passive immunoprotection.

Preferred cytotoxic T cell proliferation agents and/or NK cellproliferation agents include molecules that increase IL-2, IL-15 andIL-21 expression. Alternatively, molecules that induce IL-12 and IL-18expression are included.

The peptides of the invention are useful in treating the subject havinga disease, especially those diseases treatable by endogenous inductionof antibodies against invading pathogens or endogenous antigens ofharmful cells. The peptides of the invention can specifically be used totreat such diseases as viral infections, cancer, bacterial and yeastinfections, and/or other autoimmune diseases, which require treatmentthrough stimulation of the immune system. Such autoimmune diseasesinclude rheumatoid arthritis, psoriasis; dermatitis; systemicscleroderma and sclerosis; responses associated with inflammatory boweldisease; Crohn's disease; ulcerative colitis; respiratory distresssyndrome; adult respiratory distress syndrome (ARDS); dermatitis;meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergicconditions; eczema; asthma; conditions involving infiltration of T cellsand chronic inflammatory responses; atherosclerosis; leukocyte adhesiondeficiency; systemic lupus erythematosus (SLE); diabetes mellitus;multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergicencephalomyelitis; Sjorgen's syndrome; juvenile onset diabetes; immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T lymphocytes; tuberculosis; sarcoidosis; polymyositis;granulomatosis; vasculitis; pernicious anemia (Addison's disease);diseases involving leukocyte diapedesis; central nervous system (CNS)inflammatory disorder; multiple organ injury syndrome; hemolytic anemia;myasthenia gravis; antigen-antibody complex mediated diseases;anti-glomerular basement membrane disease; antiphospholipid syndrome;allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome;pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; Reiter'sdisease; stiff-man syndrome; Behcet disease; giant cell arteritis;immune complex nephritis; IgA nephropathy; IgM polyneuropathies;idiopathic thrombocytopenic purpura (ITP) and autoimmunethrombocytopenia.

The invention encompasses methods of substantially activating subsets oflymphocytes in a subject, in particular NK cells that attack diseasedcells directly or by antibody-dependent cellular cytotoxicity, whichcomplements activation of Fc-mediated phagocytosis, to treat a subject.In a preferred embodiment, HIV-1 replication is inhibited in the subjectby at least 50%, more preferably by at least 90% as compared to acontrol and/or levels prior to administration of the peptide in thesubject. In the presence of antibodies, inhibition may reach 100%.

In a preferred embodiment, to provide a non-specific therapeutic agentwith a relatively broad front, an agent that activates DCs, B cells, Tcells, NK and cytotoxic T cells preferably works in concert with thephagocytic cells of the immune system. The peptides of the presentinvention can accomplish this goal by concomitantly stimulating theimmune cells, including NK cells and phagocytes, and to respond inparticular to the presence of pathogen-directed antibodies. Treatmentwith the peptides of the present invention therefore preferably induceactivation of cells of the immune system in vivo and provide a sustainedendogenous defense against the pathogen.

In the following description, and for the purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the various aspects of the invention. It will beunderstood, however, by those skilled in the art, that the structures,compositions, and methods are sometimes shown or discussed generally inorder to avoid obscuring the invention. In many cases, a description ofthe material and operation is sufficient to enable one to implement thevarious forms of the invention. It should be noted that there are manydifferent and alternative technologies and treatments to which thedisclosed inventions may be applied, and the full scope of theinventions is not limited to the examples that are described below.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The term “pharmaceutically acceptable” as used herein means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which an activeingredient is administered. Such pharmaceutical carriers can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. The pharmaceutical carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. In addition, other excipients can be used.

Preferably, the subject being treated by the methods described herein isa mammal, e.g., monkey, dog, cat, horse, cow, sheep, pig, and morepreferably the subject is human.

“Effective amount” or “therapeutically effective amount” is meant todescribe an amount of therapeutic peptide or composition of the presentinvention effective to modulate the innate and adaptive immune systemsand/or treat or prevent a disease in a subject and thus produce thedesired therapeutic effect in the subject.

Typical compositions and dosage forms may comprise one or moreexcipients. Suitable excipients are well known to those skilled in theart of pharmacy, and non-limiting examples of suitable excipients areprovided herein. See, e.g., Remington's Pharmaceutical Sciences, 18thed., Mack Publishing, Easton Pa. (1990).

The present invention comprises therapeutic peptides, compositions ofthose therapeutic peptides for administration to a subject in need, andmethods to stimulate the immune system of a subject through theadministration of compositions containing those therapeutic peptides. Ingeneral, the advantage of this invention is the modulated release ofspecific cytokines and the stimulation of immune cells, including butnot limited to B cells, NK cells, CD8⁺ T cells and phagocytes, torespond to the presence of pathogen-directed antibodies. Non-limitingexamples of cytokines include immunoregulatory proteins such asinterleukins and interferons, which are secreted by cells of the immunesystem and can affect the immune response. A non-limiting example of thestimulation of immune cells is the induction of Fc-mediatedphagocytosis. An additional example is direct activation of NK cells forantibody-dependent cellular cytotoxicity. A further example isactivation of NK cells and CTL to lyse infected or cancer cells bydirect cellular cytotoxicity.

The single letter designation for amino acids is used predominatelyherein. As is well known by one of skill in the art, the single letterdesignations are as follows: A is alanine; C is cysteine; D is asparticacid; E is glutamic acid; F is phenylalanine; G is glycine; H ishistidine; I is isoleucine; K is lysine; L is leucine; M is methionine;N is asparagine; P is proline; Q is glutamine; R is arginine; S isserine; T is threonine; V is valine; W is tryptophan; Y is tyrosine.

The therapeutic peptide is preferably 5 to 8 amino acids. Preferredtherapeutic peptides are selected from the group consisting of:

VGGGS (SEQ ID NO:1) and

X1-X2-X3-X4-X5-X6-X7-X8,

wherein:

X1 is selected from the group consisting of H and N;

X2 is selected from the group consisting of P and Q;

X3 is selected from the group consisting of S and H;

X4 is selected from the group consisting of H, T, and L;

X5 is selected from the group consisting of P and K, or is absent;

X6 is selected from the group consisting of R, L and S, or is absent;

X7 is selected from the group consisting of S and L, or is absent; and

X8 is G, or is absent;

In a most preferred embodiment, the therapeutic peptide is selected fromthe group consisting of: VGGGS (SEQ ID NO:1), HPSLK (SEQ ID NO:2),NPSHPLSG (SEQ ID NO:3), NPSHPSLG (SEQ ID NO:4) and NQHTPR (SEQ ID NO:5).

Multivalent structured polypeptides comprising multiple copies of thetherapeutic peptide are preferred. In one embodiment, the multivalentstructured polypeptide comprises a construct and at least two arms, theconstruct having a central framework and each arm comprising atherapeutic peptide sequence linked to the central framework via alinker, wherein each therapeutic sequence is preferably the same.

As used herein, “construct” is defined as the entire molecule andcomprises the central framework linked with at least two arms. In apreferred embodiment, the construct comprises the central frameworklinked to 2 or more arms, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms,preferably 2 to 8 arms. In a further preferred embodiment, the constructcomprises the central framework linked to 4 arms. Each arm within theconstruct may consist of the same or different therapeutic sequenceand/or linker. In one preferred embodiment, the therapeutic sequence isthe same between arms.

The “central framework” provides a structure for attaching the arms. Thecentral framework is based on a core molecule, which has at least twofunctional groups to which molecular branches having terminal functionalgroups are bonded, e.g., a tri-lysine to which the peptide arms areadded. Such molecules may be developed or created to present a varyingnumber of branches, depending on the number of monomers branched fromthe core molecule. Each terminal functional group on each branchprovides a means of attachment to an arm. Non-limiting examples ofpreferred central framework include: ethylenediamine(1,2-ethanediamine), ethylene glycol (1,2-dihydroxyethane), polyols suchas glycerol, 3,5-diaminobenzoic acid, 1,3,5-triaminobenzene, andmonocarboxylic-diamino compounds of intermediate length. Preferably, themonocarboxylic-diamino compounds are within the range of 2 to 10 carbonsin length. Non-limiting examples of such compounds are2,3-diaminopropionic acid and 2,6-diaminocaproic acid. In a morepreferred embodiment, the monocarboxylic-diamino compound is 6 carbonsin length. Compounds that provide an aromatic central framework whichabsorbs light may be beneficial for determining peptide concentration aswell. The carboxyl group of the monocarboxylic-diamino compounds allowsthe addition of C-terminal tags including biotin derivatives. In apreferred embodiment, the central framework comprises a tri-lysine core(a lysine residue as the central molecule bonded to two lysine residues,each through its carboxyl group, to one of the amino groups of thecentral lysine residue), providing a central framework for four arms.

The “arm” comprises the therapeutic sequence, plus the linker. The“linker” comprises a peptide chain or other molecule that connects thecentral framework to the core sequence. In a preferred embodiment, thelinker comprises, but is not limited to, certain linker peptidesequences, polyethylene glycol, 6-aminocaproic acid (6-aminohexanoicacid), 8-aminooctanoic acid, and dextran. In a most preferredembodiment, the linker is GGGS (SEQ ID NO:6), GGGSGGGS (SEQ ID NO:7),SSSS (SEQ ID NO:8), SSSSSSSS (SEQ ID NO:9), or variations thereof. Thelength of the linker can be adjusted, for example, the linker GGGS (SEQID NO:6) can be repeated to provide variable lengths, e.g., repeatedtwice (GGGSGGGS (SEQ ID NO:7)), or even three or more times; additionalserine residues could be added to SSSS (SEQ ID NO:8) to also producevarying lengths of the linker. The therapeutic peptide preferablyfunctionally mimics a terminal sequence 5-acetylneuraminicacid-galactose on complex glycans, the terminal sequence being linkedα(2,3) or α(2,6). In some aspects, the therapeutic peptide functionallymimics a terminal sequence 5-acetylneuraminic acid-galactose orN-acetylgalactosamine on complex glycans, the terminal sequence beinglinked α(2,3) or α(2,6). The therapeutic peptides are advantageouslyconfigured to bind to the receptor NKG2D and/or sialic acid-bindingimmunoglobulin-like lectins and function as modulators of the immunesystem by binding to receptors on B cells, DCs, NK cells, T cells,cytotoxic T cells and/or phagocytic cells.

The therapeutic peptide is preferably administered in an amountsufficient to induce activation of NK cells in the subject and thesubject is a human. In one embodiment, the therapeutic peptide ormultivalent structured polypeptide is administered in an amountsufficient to induce antibody-mediated cellular cytotoxicity in thesubject, preferably to increase the expression of at least oneendogenous cytokine from lymphocytes elected from the group consistingof: IL-2, IL-4, IL-12, IL-16, IL-17, IL-21, TNF-β, IFN-γ and RANTESand/or decreases at least one endogenous cytokines elected from thegroup consisting of: IL-lα, IL-1β, IL-13, TNF-α.

The method may advantageously further comprise the step of administeringan antibody preparation admixed in an amount sufficient to enhanceantibody-mediated cellular cytotoxicity.

The step of determining the level of immune cells such as B cells, NKcells and/or CD8⁺ cytotoxic T cells in the subject's blood is done usingwell known methods in the art, e.g., flow cytometric analysis ofperipheral blood mononuclear cells with use of antibodies againstcell-specific surface markers. It is advantageous to further establish aratio of NK cells and/or CD8+ cytotoxic T cells compared to monocytes inthe subject's blood. In a preferred embodiment, the ratio of NK cells orCD8+ cytotoxic T cells to monocytes is 3:1 or more preferably 4:1. Thepresent invention is most effective with a higher ratio NK cells and/orCD8+ cytotoxic T cells compared to monocytes.

The present invention identifies a series of peptides that stimulateimmune response and modulate the release of specific cytokines. Thus, ina first aspect, the present invention provides a therapeutic peptideconsisting of 9 to 12 amino acids in length (including a spacersequence). In a preferred embodiment, the therapeutic peptide is in asubstantially purified form. As used herein, the term “substantiallypurified” refers to material that is substantially or essentially freefrom components which normally accompany it as found in its synthesizedstate. When the material is synthesized, the material is substantiallyor essentially free of cellular material, gel materials, culture medium,chemical precursors, contaminating polypeptides, nucleic acids,endotoxin, and other organic chemicals. Preferably, the peptide ispurified to represent greater than 90% (peptide content) of all organicmolecular species present. More preferably the peptide is purified togreater than 95% (peptide content), and most preferably the peptide ispurified to essential homogeneity, wherein other organic molecularspecies are not detected by conventional techniques. Advantageously, thetherapeutic peptide is reacted with acetic anhydride to acetylate theN-terminus of the therapeutic peptide. Acetylation protects the peptidefrom N-terminal degradation and therefore is preferred.

Scientific Basis of the Invention

Peptide sequences were identified by computer-aided molecular modelingof docking to the sugar-binding site of plant lectins, which served asreceptor analogs. The concept underlying the design of Susavion'speptides had several components. From knowledge that a number ofreceptors on cells of the immune system bind carbohydrate ligands[20,21], we focused on developing peptidic mimetics of these glycanligands. Peptides of 5 to 8 amino acids in length fill the glycanbinding site of lectins and receptors and are sufficiently short to beinvisible to the antigen-presenting processes of the immune system. Animportant aspect of the final peptide is a multivalent structure that iscapable of cross-linking receptors, an event that is critical toinitiation of a signal transduction pathway within the cell [30,31]. Todetermine the most effective amino acid sequence of a peptide, molecularmodeling was performed of docking of a single (monovalent) sequence intothe glycan-binding site of well-characterized plant lectins, which wereselected as analogs of cell-surface receptors. The crystal structures ofthe lectins were downloaded from the Protein Data Bank (PDB). ArgusLab4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, Wash.)was used for modeling. Amino acid residues that comprise the bindingsite of a lectin or receptor were selected from the literature thatdescribes each protein. Through this approach, unique peptide sequenceswere evaluated by predicted binding energy. These in silico experimentspredicted that some peptides would bind to a variety of lectins withsufficiently high affinities to encourage further characterization.

A model for interaction of the peptide designated SVH1C (SEQ ID NO:3)with the glycan-binding site of the lectin-type receptor Siglec-5(accession no. 2ZG1) is illustrated in FIG. 1. The family of siglecreceptors is specific for ligands containing terminal sialic acid (alsocalled 5-acetylneuraminic acid, Neu5Ac). Siglec-5 binds with highspecificity to glycans containing a terminal Neu5Ac(α2,8)Neu5Ac and orNeu5Ac(α2,6)Gal linkage. The predicted value for ΔG′ of −47 kJ/molcorresponds to a K_(D) of 1×10⁻⁸ M for the monovalent peptide.Cell-surface receptors that bind to these sugars include the family ofsiglecs and NKG2D, an important activating receptor on NK cells and CD8⁺cytotoxic T cells. Although NKG2D has a variety of peptide/proteinligands in vivo [17,29], the C-type lectin domain of this receptorsuggested that it may also bind to glycans. This hypothesis was affirmedwhen Imaizumi et al. [18] demonstrated that NKG2D binds glycans withspecificity for Neu5Ac(α2,3)Gal. The ligand-binding site of NKG2D(accession no. 1MPU) was constructed from data presented by Li et al.[32] and McFarland et al. [33]. Modeling predicted highly favorablebinding energy to NKG2D, with a ΔG′ of −40 kJ/mol, which correspondswith a K_(D) of about 1×10⁻⁷ M (FIG. 2).

A model for interaction of the peptide designated SV6D (SEQ ID NO:5)with the glycan-binding site of the lectin-type receptorasialylglycoprotein receptor-1 (ASGPR-1) (accession no. 1DV8) isillustrated in FIG. 3. ASGPR-1 is a homolog of CLEC10a and has a strongpreference for binding N-acetylgalactosamine over galactose [34]. Thepredicted value for ΔG′ of −40 kJ/mol corresponds to a K_(D) of 1×10⁻⁷ Mfor the monovalent peptide.

The short peptide sequence was then incorporated into multivalentstructures (FIG. 4). This design was based on the concept of avidity asa function of ligand density and entropic factors. The theoretical basisfor multivalency was provided by Mammen et al. [35], Dimick et al. [36]and Cairo et al. [37]. Multivalency should provide much more favorablebinding energy than predicted by molecular modeling. Although monovalentpeptides should be active, multivalency of ligands provides high avidityinteractions and facilitates cross-linking of receptors, which is oftenrequired for activation of cellular responses [30,31]. FIG. 5illustrates the final quadravalent structure with the active sequenceNPSHPLSG (SEQ ID NO:3). FIG. 6 illustrates the final quadravalentstructure with the active sequence NQHTPR (SEQ ID NO:5).

Direct Binding of Peptides to Lectins

The binding of SVH1C to lectins such as MAA and SNA1 [19] suggests thatthe peptide mimics Neu5Ac-Gal sequences on the termini of complexglycans. This sequence is a ligand for the receptor NKG2D on NK cellsand by T cells and CD8+ cytotoxic T cells [18]. Also, a family of 14lectin-type receptors, the siglecs (sialic acid-binding Ig-likelectins), binds Neu5Ac-Gal-sequences (reviewed in reference 13). Thesiglecs are thought to promote cell-cell interactions and regulate thefunctions of cells in the innate and adaptive immune systems throughglycan recognition. These receptors are possible targets of the peptide,as predicted by molecular modeling (FIGS. 1-3). Whereas NKG2D isspecific for the Neu5Ac(α2,3)Gal linkage, members of the siglec familyexpress specificity for the α(2,3) or α(2,6) linkages. Thus the peptideshave the flexibility to bind to all of these receptors.

Binding of SVH1C to NKG2D and Siglecs

NKG2D is not known to function as a glycan receptor in vivo, althoughthe Neu5Ac(α2,3)Gal structure binds to the C-type lectin domain of thereceptor (18). On the other hand, the siglecs have been characterized asreceptors that bind Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal [13-15]. Thesereceptors function as either inhibitory or activating when bound with aligand. Siglec-1 is expressed on macrophages and is involved in cellularadhesion but also enhances endocytosis [13]. As such, it enhancesinfection of these cells by HIV-1 by binding to glycans on the envelopof the virus [38-39]. Expression of other siglecs is distributed onother cells of the immune system [13-15].

Direct binding of SVH1C to siglecs was demonstrated by a solid-phaseassay in which recombinant chimeric siglecs were bound in microtiterwells coated with protein A/G. The chimeric siglecs contained anN-terminal glycan-binding domain and a C-terminal Fcγ domain, whichbound strongly to protein A. Biotinylated peptides were then incubatedwith the siglecs, the wells were stringently washed and the boundpeptide was detected by binding of streptavidin conjugated withperoxidase. FIG. 7 shows results of this assay with several siglecs andadditional lectin-type receptors. SVH1C bound strongly to severalsiglecs but not to CLEC9a, CLEC10a or DC-SIGN. Binding of SVH1C wasinhibited by the sialylated protein, fetuin, which indicated that SVH1Clikely binds at the glycan-binding site of siglecs. In otherexperiments, a proteomic analysis of proteins fished from PBMCs withbiotinylated SVH1C and streptavidin-agarose identified Siglec-15, anactivating receptor found on myeloid cells (40), among the complex ofproteins that bound to the peptide. Among myeloid cells that expressSiglec-15 are macrophages and dendritic cells [13].

The solid-phase assay was also used to determine binding of SVH1C toNKG2D (FIG. 8). Fc-chimeric NKG2D was bound in microtiter wells coatedwith protein A/G, which binds strongly to the Fc domain. Binding ofbiotinylated svH1C was measured by activity of peroxidase conjugated tostreptavidin. Strong binding was observed, with a KD of approximately 1μM. As shown in FIG. 8, binding of the peptide was inhibited to thefetuin and the trisaccharide sialyllactose, which indicated that thepeptide bound in the glycan-binding site on the receptor. Theglycoprotein fetuin is an effective probe to confirm binding of thepeptide in the glycan-binding site of the receptors. Each molecule offetuin contains collectively 12 to 15 oligosaccharides that terminatepredominantly as Neu5Ac-Gal, with nearly equal α(2,3) and α(2,6)linkages, on three N-linked and three O-linked glycans [41,42].

Among the siglec receptors, most are inhibitory receptors and contain anITIM (immunoreceptor tyrosine-based inhibitory motif) within theircytosolic domain, whereas a few, in particular Siglec-14, Siglec-15, andSiglec-16 function with a cytoplasmic activating adaptor protein, DAP12[13,40]. NKG2D is also an activating receptor and functions inassociation with the cytoplasmic, adaptor proteins DAP10 and DAP12,which contain an ITAM (immunoreceptor tyrosine-based activation motif)[17,29]. The function of these receptors is regulated by phosphorylationof the tyrosine residue within the regulatory motif. As illustrated inFIG. 9, treatment of human peripheral blood mononuclear cells (PBMCs)with 100 nM SVH1C for 5 min caused dramatic changes in thephosphorylation state of several receptors. Phosphorylated inhibitoryreceptors commonly function by recruiting SHP-1, a phosphatase thatinactivates other receptors [13-15]. Thus, dephosphorylation of thesereceptors attenuates their activity.

In Vivo Proliferation of Immune Cells

To determine whether a decrease in activity of inhibitory receptors isreflected by stimulation of proliferation of immune cells in vivo, SVH1C(SEQ ID NO:3) was injected subcutaneously every other day at a dose of 1nanomole per gram body weight and populations of immune cells in theperitoneal cavity were measured by flow cytometry. Injections wereadministered on day 0, 2 and 4, and peritoneal lavage was performed toobtain immune cells. Cells from three animals at each time point werepooled and analyzed by flow cytometry. As illustrated in FIG. 10, mostcells types proliferated over the period of treatment. In particular,DCs (CD11c⁺), NK cells (NK1.1⁺), CD3⁺, CD4⁺ and CD8⁺ T cells, and Bcells (CD19⁺) populations increased several-fold, including those thatexpressed the activation marker CD69⁺. In particular, memory B cells,which express CD73, CD80 and CD273, increased significantly. The numberof immune cells increased by at least 1.5 fold, at least 2 folds, atleast 2.5 folds, at least 3 folds, at least 3.5 folds, at least 4 folds,at least 4.5 folds, or at least 5 folds with the administration of SVH1C(SEQ ID NO:3) compared to the number of immune cells prior to thetreatment.

In a test of the activity of SVH1C to inhibit replication of viralreplication, an experiment was conducted with PBMCs in which SV6B (SEQID NO:2) and SVH1C (SEQ ID NO:3) were added to cultures to which HIV-1was added at a low titer. It was found that when the cultures wereoverwhelmed with a high input of the virus, the percent ofneutralization was reduced. Thus, in subsequent experiments the viralinput was reduced to about 30 TCID50. An assay was performed in whichpeptides were added to the culture without antiserum. For the resultsshown in FIG. 11, the peptides alone inhibited viral replication by 80to 90%. IC50 values in this experiment were 2 μM for SVH1C and about 300μM for SV6B. Because antibodies were not present in this experiment,antibody-mediated phagocytosis did not contribute significantly toneutralization. Flow cytometric analysis of the PBMCs indicated arelatively high NK/monocyte ratio in these cultures.

Induction of Cytokine Release

To determine whether activation of cells by the peptides could bedetected by induction of release of cytokines, cultured peripheral bloodmononuclear cells (PBMCs) were treated with one peptide embodiment ofthe present invention and, after 4 h incubation, the medium wascollected and assayed for changes in the amounts of 40 differentcytokines. A therapeutic peptide construct containing four copies of thecore sequence VGGGS (SEQ ID NO:1), HPSLK (SEQ ID NO:2) or NSPHPLSG (SEQID NO:3) was added at a concentration of 100 nM in each of the assays.Approximately 3 million cells of frozen human PBMCs were thawed at 37°C. and transferred to a 50 mL conical tube where 8 mL of wash mediumwere added slowly. Then an additional 8 ml of wash medium were added andswirled to mix. The cells were then centrifuged at 330 g for 10 min, thesupernatant was removed and the pellet was resuspended in 10 mL washmedium and centrifuged as above. The washed cells were then resuspendedin RPMI-1640 medium containing 10% FBS to about 6 million cells per mLand 100 mL of the suspension were added into each well of a 96-wellmicrotiter plate and incubated overnight at 37° C. in humidified 5% CO₂.After 24 h the medium was replaced with 200 μL fresh RPMI-1640 mediumcontaining 10% FBS and incubated at 37° C. in humidified 5% CO₂ for 2days. The peptide aliquot was then added to samples in duplicate at afinal concentration of 100 nM and incubated at 37° C. in humidified 5%CO₂ for 4 h. The medium was then removed and stored at −80° C. Thesamples were analyzed for production of cytokines. One set of controlcells was not treated with an experimental agent. A second set ofcontrol cells was treated with lipopolysaccharide, an agent commonlyused to induce production of a variety of inflammatory cytokines. Thepositive control for inflammation was essential to ensure that thepeptides did not produce an unregulated inflammatory response.

Culture medium was removed for assay of cytokine levels with methodsdeveloped by RayBiotech, Inc. (Norcross, Ga.). In this technology,membrane arrays of antibodies against cytokines are incubated withsamples of media. After washing, the array was incubated with a cocktailof all antibodies tagged with biotin. The membrane was then washed freeof unbound antibodies and incubated with streptavidin, labeled with afluorescent dye, which binds to biotin. After a final wash, the membranearrays were read in a fluorescence detector.

The peptides stimulated release of several important cytokines. Inparticular, IL-21, a cytokine produced by CD4+ T cells that is requiredfor proliferation and differentiation of natural killer cells and CD8+cytotoxic lymphocytes. Additional cytokines released by the generalpopulation of T cells in response to treatment with the peptides of thisinvention were IFN-γ, IL-4, IL-8, IL-16, IL-17, TNF-β, and RANTES. Ofimportance, release of the inflammatory cytokines IL-1α, IL-1β, IL-6,IL-10, and TNF-α were not induced. Release of other important cytokines,notably Eotaxin-2, IL-10, and IL-13, was reduced (Table 1).

TABLE 1 Release of cytokines by PBMC cultures. Cytokine Source ActivityIncreased IL-8 Macrophages Activation of neutrophils IL-16 T cellsLymphocyte chemoattractant IL-17 T cells Stimulates secretion of IL-6,IL-8, G-CSF IL-21 T cells Mediates innate and adaptive immune responses,affects all lymphocytes, dendritic cells and monocytes IFN-γ NK cellsAnti-viral, immunoregulatory, anti-tumor properties TNF-β T cellsCytolytic or cytostatic for many tumors MIP-1d T, B, NK cells Macrophageinflammatory protein, activates dendritic cells, granulocytes, inducessynthesis of pro- inflammatory monocytes cytokines RANTES T cellsChemotactic for T cells, eosinophils and basophils Decreased Eotaxin-2Dendritic cells, Chemotaxis of eosinophils, basophils (inflammatory)monocytes IL-10 Monocytes, Inhibits synthesis of IFN-γ, IL-2 and TNF-βmacrophages IL-13 T cells Downregulates inflammatory cytokines

The mixture of cytokines released from PBMCs, in particular T cells, inresponse to the peptides described herein should provide, either inisolation or in combination with other treatments, an effectivemodulation of the immune system. Treatment with the peptides of thepresent invention should induce activation of cells of the immune systemin vivo and provide a sustained endogenous elevation of beneficialcytokines, in contrast to the rapid disappearance of these proteins whengiven exogenously. These cytokine responses are presumably in additionto direct activation of the immune cells engaged in fighting a disease.

The embodiments and examples set forth herein were presented in order tobest explain the present invention and its practical application and tothereby enable those of ordinary skill in the art to make and use theinvention. However, those of ordinary skill in the art will recognizethat the foregoing description and examples have been presented for thepurposes of illustration and example only. The description as set forthis not intended to be exhaustive or to limit the invention to theprecise form disclosed. Many modifications and variations are possiblein light of the teachings above without departing from the spirit andscope of the forthcoming claims. Although the examples herein disclosethe therapeutic efficacy of the peptides of the present invention, withrespect to neutralizing replication of the HIV virus, for example, thepeptides should be useful to treat a wide variety of infections ordisorders, including prophylactic treatments for prevention of suchmaladies, and for enhancing or stabilizing the well-being of healthysubjects.

Similar experiments were performed to show that SV6D (SEQ ID NO:5) alsobinds to lectin-type receptors but to a different set as compared withSVH1C (SEQ ID NO:3). Direct binding of SV6D to receptors specific forN-acetylgalactosamine or galactose was demonstrated by a solid-phaseassay in which recombinant receptors containing a poly-histidine tagwere bound in microtiter wells coated with Nickel. Biotinylated peptideswere then incubated with the receptors, the wells were stringentlywashed and the bound peptide was detected by binding of streptavidinconjugated with peroxidase. FIG. 12 shows results of this assay withseveral lectin-type receptors. SV6D bound strongly to CLEC10a, langerin,ASGPR-1 and dectin-1 but not to CLEC9a, DC-SIGN or Siglec-1.

To determine whether binding of SV6D to cell-surface receptors wouldtransfer signals to the cytoplasm of cells, cultures of PBMCs wereincubated with 100 nM SV6D and samples were removed after 5, 10 or 15minutes and compared with the phosphorylation state of signaltransduction kinase intermediates in untreated cells. As illustrated inFIGS. 13A and 13B, the phosphorylated form of several intermediateschanged rapidly and dramatically. In particular, the phosphorylatedforms of proteins p53, CREB and PRAS40 were markedly decreased at 10minutes but rebounded by 15 minutes. These rapid changes may representremodeling of the phosphor-proteins.

In other experiments, the activity of SV6D (SEQ ID NO:5) to stimulateproliferation of immune cells in vivo was determined. SV6D was injectedsubcutaneously every other day at a dose of 1 nanomole per gram bodyweight and populations of immune cells in the peritoneal cavity weremeasured by flow cytometry. Injections were administered on day 0, 2 and4, and peritoneal lavage was performed to obtain immune cells. Cellsfrom three animals at each time point were pooled and analyzed by flowcytometry. As illustrated in FIG. 14, most cells types proliferated overthe period of treatment. In particular, DCs (CD11c⁺), NK cells (NK1.1⁺),CD3⁺, CD4⁺ and CD8⁺ T cells, and B cells (CD19⁺) populations increasedseveral-fold, including those that expressed the activation markerCD69⁺.

A particularly difficult to treat cancer in women is ovarian cancer. Alate-stage condition of ovarian cancer is accumulation of ascites in theperitoneal cavity. In a mouse model of ovarian cancer, a cancer cellline was implanted into the peritoneal cavity. The cells form tumors andin time also release malignant cells into the cavity. The activity ofSV6D to treat ovarian cancer was tested by alternate-day injections of 1and 0.1 nanomole per gram of SV6D. Development of ascites was monitoredby an increase in body weight of the animals. Ascites accumulation wasevident at the start of treatment and the weights shown in FIG. 15 areafter two weeks of treatment. The effectiveness of the 0.1 nmole/g dosewas similar to that of the drug paclitaxel, the currently usedchemotherapeutic drug.

These experiments showed that SVH1C and SV6D, although expressing verydifferent activities in binding to receptors in vitro, had remarkablysimilar effects in stimulation of proliferation and activation ofperitoneal immune cells in vivo. SVH1C has strong antiviral activity(FIG. 11) and SV6D shows strong potential in inhibiting growth of tumorcells (FIG. 15). Therefore, these peptides approach enhancement ofimmune system activity from different angles, which makes thecombination of these peptides a potentially powerful therapeuticembodiment. In other embodiments, SV6D may be used in combination withother therapeutic peptides with predominate antiviral activity, such asthe peptides set having the amino acid sequence VGGGS (SEQ ID NO:1),HPSLK (SEQ ID NO:2), NPSHPSLG (SEQ ID NO:4), HPSLG (SEQ ID NO:10), HPSLL(SEQ ID NO:11), HPSLA (SEQ ID NO:12).

Toxicity of Peptides

Human PBMCs were incubated 3 days with peptides and then assay plateswere stained with the soluble tetrazolium-based dye MTS to determinecell viability. The mitochondrial enzymes of metabolically active cellsmetabolize MTS to yield a colored formazan product. After an incubationperiod of 4 to 6 h at 37° C., the plates were readspectrophotometrically. Cells treated with peptide SVH1C alone or withdiluted anti-HIV antiserum were 100±2% viable at peptide concentrationsof 1 nM to 1 μM. In another assay to determine cytotoxicity, cells weredoubly stained with acridine orange and ethidium bromide. In this assay,viable cells fluoresce green while dead cells fluoresce red. SVH1C didnot exhibit cytotoxicity at a concentration of 1 mM, a concentration10⁶-fold greater than an effective bioactive concentration of 1 nM [43].

Toxicity of the peptide in vivo was tested by injection of a peptideinto animals. In preliminary studies on rats, intravenous injections ofpeptides that provided 1000-fold greater concentrations than an expectedtherapeutic dose was well tolerated by the animals and no adverseeffects of the peptide were been observed. The peptides can beadministered in a number of ways, including without limitation byinjection (intravenously, subcutaneously, intramuscularly orintraperitoneally, topically (transmucosally, transbuccally,sublingually, or transdermally) and/or orally (liquid, tablet orcapsule).

CONCLUSION

The data shown herein demonstrate that the peptide SVH1C (NPSHPLSG, SEQID NO:3), functionally mimic glycans with terminal Neu5Ac-Gal sequences.Receptors such as NKG2D and siglecs bind these glycans. Siglec-1 isexpressed on monocytes and macrophages and is involved in cellularadhesion but also enhances endocytosis of viruses [38,39]. Therefore,these peptides should function as modulators of cell activity by servingas a ligand for these receptors.

Similarly, the data shown herein demonstrate that the peptide SV6D(NQHTPR, SEQ ID NO:5), functionally mimic glycans with terminalN-acetylgalactosamine or galactose residues. Receptors such as CLEC10aand ASGPR-1 bind these glycans. CLEC10a is expressed on immaturedendritic cells and macrophages and enhances endocytosis [22,24].Therefore, these peptides should function as modulators of cell activityby serving as a ligand for these receptors.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols and materials described as these canvary. It is also understood that the terminology used herein is for thepurposes of describing particular embodiments only and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

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1. (canceled)
 2. A method of treating an immunocompromised subject, asubject having cancer and/or a subject having a viral infection: themethod comprising administering to the subject in need thereof atherapeutic peptide or a multivalent structured polypeptide comprisingmultiple copies of the therapeutic peptide, the therapeutic peptideconsisting of 5 to 8 amino acids and selected from the group consistingof: VGGGS (SEQ ID NO:1) and X1-X2-X3-X4-X5-X6-X7-X8, wherein X1 isselected from the group consisting of H and N; X2 is selected from thegroup consisting of P and Q; X3 is selected from the group consisting ofS and H; X4 is selected from the group consisting of H, T, and L; X5 isselected from the group consisting of P and K, or is absent; X6 isselected from the group consisting of R, L and S, or is absent; X7 isselected from the group consisting of S and L, or is absent; and X8 isG, or is absent; wherein the therapeutic peptide or multivalentstructured polypeptide is in an amount sufficient to increaseproliferation of immune cells in the subject.
 3. The method of claim 2,wherein the therapeutic peptide is selected from the group consistingof: VGGGS (SEQ ID NO:1), HPSLK (SEQ ID NO:2), NPSHPLSG (SEQ ID NO:3),NPSHPSLG (SEQ ID NO:4), and NQHTPR (SEQ ID NO:5).
 4. The method of anyone of claim 2, wherein the subject has cancer.
 5. The method of claim2, wherein the subject has cancer and the therapeutic peptide is NQHTPR(SEQ ID NO:5), or the multivalent structured polypeptide comprisesmultiple copies of the therapeutic peptide, wherein the therapeuticpeptide or multivalent structured polypeptide is administered in anamount sufficient to treat the cancer in the subject.
 6. The method ofclaim 2, wherein the multivalent structured polypeptide is administeredto the subject and is branched.
 7. The method of claim 2, wherein thesubject has a persistent viral infection.
 8. The method of claim 7,wherein the persistent viral infection is selected from the groupconsisting of: an HIV/AIDS infection, a CMV infection, a HBV infection,and a HCV infection.
 9. The method of claim 2, wherein the subject hasan HBV infection.
 10. The method of claim 2, wherein the therapeuticpeptide or multivalent structured polypeptide stimulates proliferationof immune cells selected from the group consisting of macrophages;dendritic cells; natural killer cells; natural killer T cells; CD3⁺,CD4⁺ and CD8⁺ T cells; B cells; and combinations thereof; and the methodfurther comprises the steps of determining (a) the level of immune cellsin the subject before administering the composition; and (b) the levelof immune cells in the subject after administering the composition. 11.(canceled)
 12. The method of claim 10, wherein (a) and (b) aredetermined with flow cytometry; and the ratio of (b) to (a) is at least1.5. 13-14. (canceled)
 15. The method of claim 2, wherein thetherapeutic peptide or multivalent structured polypeptide activates theimmune cells by binding to at least one activating receptor selectedfrom the group consisting of: NKG2D and CLEC10a.
 16. The method of claim2, further comprising administering to the subject a second therapeuticpeptide or a multivalent structured polypeptide comprising multiplecopies of the second therapeutic peptide, wherein the multivalentstructured polypeptide is branched. 17-19. (canceled)
 20. The method ofclaim 16, wherein the second therapeutic peptide functionally mimics aterminal sequence 5-acetylneuraminic acid-galactose orN-acetylgalactosamine on complex glycans, the terminal sequence beinglinked α(2,3) or α(2,6) or terminal sugars such as N-acetylgalactosamineor galactose.
 21. The method of claim 2, wherein the therapeutic peptideor multivalent structured polypeptide is in a composition comprising acarrier.
 22. The method of claim 21, wherein the composition furthercomprises at least one agent selected from the group consisting of: ananti-inflammatory agent, a cytotoxic T cell proliferation agent, or a NKcell proliferation agent; and a therapeutic peptide or a multivalentstructured polypeptides of the invention.
 23. The method of claim 21,wherein the composition further comprises an antibody preparationadmixed in an amount sufficient to enhance antibody-mediated cellularcytotoxicity in a subject; or an immunoglobulin admixed therewith in anamount sufficient to enhance passive immunoprotection in the subject.24. A therapeutic composition comprising a carrier, a therapeuticpeptide or a multivalent structured polypeptide comprising multiplecopies of the therapeutic peptide, wherein the therapeutic peptide isNQHTPR (SEQ ID NO:5), wherein the therapeutic peptide or multivalentstructured polypeptide is in an amount sufficient to treat the cancer ina subject; and at least one agent selected from the group consisting of:a B cell proliferative agent, a cytotoxic T cell proliferation agent, ora NK cell proliferation agent.
 25. The therapeutic composition of claim24, further comprising an antibody preparation admixed in an amountsufficient to enhance antibody-mediated cellular cytotoxicity in asubject; or further comprises an immunoglobulin admixed with thepolypeptide composition in an amount sufficient to enhance passiveimmunoprotection.
 26. The therapeutic composition of claim 25, furthercomprising a second therapeutic peptide or a multivalent structuredpolypeptide comprising multiple copies of the second therapeuticpeptide, wherein the multivalent structured polypeptide is branched.27-29. (canceled)